| Objective: Angiogenin(ANG)is one of the early found angiogenic factors,which can promote angiogenesis,regulate new angiogenesis and maintain vascular stability,and necessary permissive factor for other angiogenic factors include vascular endothelial growth factor(VEGF)and other important angiogenic factors playing a role in promoting vascular growth.ANG also exists in human tissues and body fluids,such as plasma,serum,amniotic fluid and follicular fluid,which may participate in a variety of physiological and pathological processes.Moreover,in 2016,ANG was newly discovered to play a role as stem cell regulator in blood stem cells,skin stem cells and tumor stem cells.ANG also has different cell-specific functions,which makes the original stem cells tend to be static but promotes the proliferation of precursor stem cells.Therefore,it has a very broad therapeutic potential in transplantation,tissue repairment and stem cell application engineering.ANG can promote the proliferation of both vascular endothelial cells and cancer cells.In addition,ANG was found in amniotic fluid and follicular fluid,we speculate that ANG may play a role in the occurrence and development of female reproductive diseases.During the breeding C57BL/6J mice,we found that the reproductive capacity of Ang gene knockout mice(Ang(-/-))was significantly lower than that of wild type(WT)mice(Ang(+/+)).It was shown that the fertility rate and litter size of mice decreased.Therefore,in-depth study of the relationship between ANG and female reproduction would provide a new idea for the diagnosis and treatment of female reproductive related diseases.The purpose of this study is to explore the effects of Ang gene knockout on the ovarian function of mice from the aspects of fertility rate,litter size,estrous cycle,ovarian tissue morphology and follicle number in different periods,so as to lay a foundation for the study of the role and mechanism of ANG in the field of reproduction and its future development and application in the field of reproduction.The paper is divided into two parts,as follows:Part Ⅰ To investigate the effect of Ang gene knockout on the ovarian function of miceMethod: Two C57BL/6J Ang gene knockout homozygous Ang(-/-)and two heterozygous Ang(+/-)female mice,donated by Professor Xu Zhengping of Zhejiang University Medical College,were used in this experiment.They were bred in our experimental center and further identified according to the method of Hu Guofu Laboratory of Tufts University in the United States.The ovarian function of Ang(-/-)and WT mice was observed and compared from the following three aspects:1)In each group,20 8-week-old female mice were caged together for one week continuously according to the ratio of male and female 1:1,then the male mice were taken out,the female mice were continuously observed for another 21 days,and the average pregnancy rate and the average number of offspring per fetus of the two groups were counted.2)8-week-old female mice,3 in each group,the estrous cycle of each mouse was observed by vaginal smear method for continuous 35 days.3)12 female mice in each group.3 rats were killed at 4,6,8,12 weeks of age at each stage.Then the abdominal cavity was cut open,and the ovarian tissue was fixed,paraffin embedded and sectioned.The morphology of the ovarian tissue was observed by HE staining,and the number of follicles at all levels in the ovarian tissue was counted and statistically analyzed.Result:1.Fertility ability observation: The results showed that the pregnancy rate and the average number of offspring per fetus of Ang(-/-)female mice decreased.The pregnancy rate of Ang(-/-)group 40.00% was lower than that of WT group 47.50%,but there was no markedly difference(P=0.342).In Ang(-/-)group,the average number per fetus was 4.55±0.94,which was markedly lower than that in WT group 7.20±1.15(P<0.001).2.Observation of estrous cycle by vaginal smear: The results showed that the estrus cycle of female Ang(-/-)mice was disordered.The average estrus period of WT group was 8.33±1.03 days,and that of Ang(-/-)group was 1.00±0.89 days.The estrus period of Ang(-/-)group was markedly shorter than that of WT group(P<0.001).The average oestrus interval of WT group was 6.17±0.75 days,and that of Ang(-/-)group was 12.5±1.38 days,which was markedly longer than that of WT group(P<0.002).The average preestrus time of WT group was 4.00±0.63 days,and that of Ang(-/-)group was 8.83 ± 1.17 days,which was markedly longer than that of WT group(P=0.003).There was no markedly difference between the two groups in the period of metestrus.3.Morphological observation of ovary: There was no markedly difference in ovarian tissue of the two groups at the age of 4 and 6 weeks.At the age of 8 and 12 weeks,the ovarian tissue structure of Ang(-/-)mice was disordered,the follicles of cortical atresia were obviously visible,the ovarian stroma was proliferated and thickened,and the arrangement of granulosa cells of corpus luteum was loose and irregular.4.Comparison of follicle numbers at different levels in ovaries of 4,6,8 and 12 week mice1)Primordial follicles number(1)4W: The average number of primordial follicles in WT group was 125.33±11.33 and that in Ang(-/-)group was 119.83±2.79,(P=0.137).(2)6W: The average number of primordial follicles in WT group was 118.23±10.36 and that in Ang(-/-)group was 115.00±5.87(P=0.355).(3)8W: The average number of primordial follicles in WT group was 64.00±5.22 and that in Ang(-/-)group was62.33±5.54(P=0.793).(4)12W: The average number of primordial follicles in WT group was 33.17±6.08 and that in Ang(-/-)group was 27.43±5.38(P=0.979).There was no obvious difference in the average number of primordial follicles in each stage between the two groups.2)Primary follicles number(1)4W: The average number of primary follicles in WT group was 26.33±5.32 and that in the Ang(-/-)group(15.50±3.83)was obviously less(P<0.001).(2)6W:The average number of primary follicles in WT group was18.33±3.61 and that in the Ang(-/-)group(9.33±3.01)was obviously less(P<0.001).(3)8W: Ang(-/-)group(9.33±3.67)was lower than WT group(13.17±2.32),but both had no significant difference(P=0.401).(4)12W: Ang(-/-)group(7.50±2.07)was lower than WT group(9.50±2.17),and no obvious difference between the two.(P=0.126).3)Secondary follicles number(1)4W: The average number of primary follicles in WT group was 22.67±3.01 and that in the Ang(-/-)group(14.83±3.06)was markedly less(P<0.001).(2)6W: The average number of primary follicles in WT group was11.73±2.48 that in the Ang(-/-)group(4.53±1.47)was markedly less(P<0.001).(3)8W:The average number of primary follicles in WT group was 11.33±2.73 and that in the Ang(-/-)group(5.50±1.86)was markedly less(P<0.001).(4)12W: Ang(-/-)group(3.87±1.47)was lower than WT group(6.50±1.87),and no obvious difference between the two.(P=0.496).4)Antral follicles number(1)The average number of primary follicles in WT group was 13.50±2.59,and that in Ang(-/-)group was 9.17±1.47,which was obviously less than that in WT group(P=0.0051).(2)6W: Ang(-/-)group(9.33±2.07)was lower than WT group(11.50±2.59),but both had no significant difference(P=0.493).(3)8W:Ang(-/-)group(6.66±2.42)was lower than WT group(8.67±1.75),but both had no significant difference(P=0.275).(4)12W: There was no apparently difference between WT group 7.17±2.15 and Ang(-/-)group 6.15±1.72(P=0.638).5)Atretic follicles number(1)4W: The average number of primary follicles in WT group was 10.50±2.66 and that in Ang(-/-)group was 28.50±2.59,which was apparently higher than that in WT group(P<0.001).(2)6W: Ang(-/-)group(42.64±6.19)was higher than WT group(36.67±3.83),but both had no markedly difference(P=0.071).(3)8W: The average number of primary follicles in WT group was41.17±3.76 and that in Ang(-/-)group was 50.68±4.41,which was obviously higher than that in WT group(P=0.003).(4)12W: Ang(-/-)group(62.15±8.33)was higher than WT group(53.33±5.72),and no obvious difference between the two.(P=0.058).6)Corpus luteum number(1)4W: Ang(-/-)group(6.17±2.14)was lower than WT group(7.00±1.41),but both had no markedly difference(P=0.406).(2)6W: There was no significant difference between WT group(10.50±1.87)and Ang(-/-)group(8.69±2.16)was lower than WT group(10.50±1.87),and no markedly difference between the two.(P=0.147).(3)8W: The average number of primary follicles in WT group was 13.85±2.64 and that in Ang(-/-)group was 5.00±1.67,which was markedly less than that in WT group(P<0.001).(4)12W: The average number of primary follicles in WT group was 8.13±2.93 and that in Ang(-/-)group was 4.50±2.07,which was obviously less than that in WT group(P=0.031).Conclusion: In conclusion,the results showed that C57BL/6J female mice of Ang gene knockout,the fertility was significantly lower than that of normal WT mice,the number of offspring per fetus was significantly decreased,the estrus cycle are the disordered with obvious decreasing of the estrus period,and the prolongation of the pre and inter estrus periods.It affects the development and maturation of follicles at all stages,but the most obvious at early stage of 4W,as shown the number of growth follicles including primary,secondary and antral follicles decreased significantly at4 W,but did not affect ovulation,because corpus luteum number did not decrease significantly.It can promote follicular atresia,which is manifested by the obvious increase of atresia follicles at 4 and 8 W,affected on late ovulation at 8 and 12 W,because there was no significant difference in antral follicles,but the number of corpus luteum decreased significantly.The results also show that the ovarian tissue structure was disordered,the follicles of cortical atresia were obvious,the ovarian stroma was hyperplasia and thickening,the arrangement of luteal granulosa cells was loose and irregular at 8 and 12 W.The results proved that Ang gene knockout had significant effect on the ovarian function of mice.Part Ⅱ Effect of Angiogenin gene knockout on cyclophosphamide in duced premature ovarian failureIn the first part,it has proved that Ang gene knockout has a significant effect on the ovarian function of mice.This part design to further understand whether Ang gene knockout has an impact on ovarian function under pathological conditions.Chemotherapy-induced premature ovarian failure(POF)or ovarian dysfunction often occur.In recent years,chemotherapy drugs such as cyclophosphamide(CTX)and other damage to ovarian function have attracted widespread attention.CTX damage ovarian function is also an important factor in POF.Therefore,it is urgent and necessary to study the protective treatment of ovarian function damage caused by chemotherapy drugs.In this part,CTX was used to challenge the ovarian function of Ang knockout mice to observe whether Ang knockout will aggravate or alleviate POF,so as to further understand the impact of Ang gene knockout on the ovarian function under pathological conditions,and will possibly provide new ideas for the diagnosis and treatment of POF.Method: Separate the 8-week-old C57BL/6J female mice into three groups:normal group(WT),CTX group(WT+CTX),Ang(-/-)+CTX group(6 mice in each group).The mice in CTX group and Ang(-/-)+CTX group received POF modeling method,that is,at the first day,intraperitoneal injection of load dose CTX50 mg.kg-1.d-1,and the second day,intraperitoneal injection of maintenance dose of8 mg.kg-1.d-1,continuous intraperitoneal injection for 14 days.The normal mice were injected with the same volume of normal saline at the corresponding time,and general condition of the mice was observed and recorded for 24 hours after the last administration,the mice eyes were plucked for blood,serum was separated and stored at-80 ℃,then mice were killed by dislocation off the neck,and then weighed, ovaries were taken out,the wet weight of ovaries weighed,the ovarian index calculated,the left ovary fixed,paraffin embedded,sectioned,stained and the morphology of ovaries observed.The numbers of follicles at all levels were observed and counted,and the percentage counted.The right ovarian tissue was fixed,paraffin embedded,sectioned,and the degree of ovarian fibrosis observed by VG staining.The serum estradiol(E2),follicle stimulating hormone(FSH)and anti Muller tube hormones(AMH)were measured by enzyme-linked immunosorbent assay in the later period.The results are as follows:Result:1.Ovarian weight and ovarian index1)Ovarian weight comparison: normal group(0.0024±0.0004)g,CTX group(0.0010±0.0003)g,Ang(-/-)+CTX group(0.0007±0.0003)g,CTX group was markedly lower than WT group(P<0.001),but Ang(-/-)+CTX group and CTX group had no markedly difference(P=0.152).2)Ovarian index comparison: normal group 0.0107±0.0007,CTX group0.0044±0.0009,Ang(-/-)+CTX group 0.0038±0.0017,CTX group significantly lower than WT group(P<0.001),but Ang(-/-)+CTX group and CTX group had no markedly difference(P=0.342).2.Comparison of follicle count at different levels1)Primordial follicles: normal group 50.13±8.79,CTX group 29.75±5.20,Ang(-/-)+CTX group 9.00±3.34,CTX group was markedly lower than WT group(P<0.001),Ang(-/-)+CTX group was markedly lower than CTX group(P<0.001).2)Primary follicles: normal group 13.13±3.60,CTX group 9.25±2.38,Ang(-/-)+CTX group 4.25±1.16,CTX group significantly was lower than WT group(P=0.007),Ang(-/-)+CTX group also was markedly lower than CTX group(P=0.001).3)Secondary follicles: normal group 11.25±4.40,CTX group 8.50±2.20,Ang(-/-)+CTX group 4.50±1.31,CTX group was significantly lower than WT group(P=0.075),Ang(-/-)+CTX group was obviously less than CTX group(P=0.013).4)Antral follicles: normal group 6.63±2.07,CTX group 2.13±1.81,Ang(-/-)group1.25±1.16,CTX group was significantly lower than WT group(P<0.001).But no markedly difference between Ang(-/-)+CTX group and CTX group(P=0.321).5)Atretic follicles: normal group 15.75±3.45,CTX group 33.88±8.79,Ang(-/-)+CTX group 42.50±7.54,CTX group was obviously higher than WT group(P<0.001),Ang(-/-)+CTX group was significantly higher than CTX group(P=0.002).6)Corpus luteum: normal group 7.50±2.56,CTX group 3.25±2.25,Ang(-/-)+CTX group 1.75±1.58,CTX group was significantly lower than WT group(P=0.001),But Ang(-/-)+CTX group and CTX group had no markedly difference(P=0.182).3.Comparison of the total number of follicles and the proportion of follicles at all levels:1)Comparison of total follicles: normal group 104.38±16.03,CTX group86.75±8.63,Ang(-/-)+CTX group 63.25.±20.41,CTX group was significantly lower than WT group(P=0.006)and Ang(-/-)+CTX group was significantly lower than CTX group(P=0.001).2)Proportion of primordial follicles: normal group(47.93±2.88)%,CTX group(34.26±4.86)%,Ang(-/-)+CTX group(14.15±4.37)%,CTX group was significantly lower than WT group(P<0.001),Ang(-/-)+CTX group was significantly lower than CTX group(P<0.001).3)Proportion of primary follicles: normal group(12.51±2.73)%,CTX group(10.78±3.13)%,Ang(-/-)+CTX group(6.70±1.41)%,CTX group was lower than normal group and both had no markedly difference(P=0.188),but Ang(-/-)+CTX group was markedly lower than CTX group(P=0.004).4)Proportion of secondary follicles:normal group(10.63±3.28)%,CTX group(9.81±2.40)%,Ang(-/-)+CTX group(7.21±2.41)%,CTX group was lower than normal group and both had no markedly difference(P=0.551),nor between Ang(-/-)+CTX group and CTX group(P=0.071).5)Proportion of antral follicles: normal group(6.24±1.19)%,CTX group(2.52±2.15)%,Ang(-/-)+CTX group(1.87±1.71)%,CTX group was lower than normal group and both had markedly difference(P<0.001),but no markedly difference between CTX group and Ang(-/-)+CTX group(P=0.461).6)Proportion of atretic follicles: normal group(15.41±4.14)%,CTX(38.76±7.79)%,Ang(-/-)+CTX group(67.21±8.79)%,CTX group was obviously higher than normal group(P=0.022)and Ang(-/-)+CTX group was significantly higher than CTX group(P<0.001).7)Proportion of corpus luteum: normal group(7.28±2.47)%,CTX group(3.87±2.99)%,Ang(-/-)+CTX group(2.86±2.81)%,CTX group was obviously lower than normal group(P<0.001),but CTX group and Ang(-/-)+CTX group had no markedly difference(P=0.472).4.Test results of sex hormone level:1)AMH: normal group(7.36±0.55)pg/ml,CTX group(2.85±0.3)pg/ml,Ang(-/-)+CTXgroup(1.70±0.63)pg/ml,CTX group was markedly lower than normal group(P<0.001),so was Ang(-/-)+CTX group and CTX group(P<0.001).2)E2:normal group(35.99±1.13)pg/ml,CTX group(23.81±1.03)pg/ml,Ang(-/-)+CTX group(18.85±1.06)pg/ml,CTX group was markedly lower than normal group(P<0.001),so was Ang(-/-)+CTX group and CTX group(P<0.001).3)FSH:normal group(2.35±0.41)m IU/ml,CTX group(3.42±0.34)m IU/ml,Ang(-/-)+CTX group(6.08±0.33)m IU/ml,CTX group was markedly lower than normal group(P<0.001),so was Ang(-/-)+CTX group and CTX group(P<0.001).5.Histological observation of ovary Compared with the normal group,the ovarian tissue in CTX group was obviously atrophic,the tissue structure was disordered and interstitial necrosis with fibrosis.There were more atretic follicles and follicular necrosis in cortex;compared with the CTX group,the ovarian tissue of the Ang(-/-)+CTX group was more atrophic and disordered,with a large number of atretic follicles and necrosis of follicles.The growth follicles were rare,and the degree of interstitial fibrosis was significantly increased.Conclusion: The results show that compared with the normal female mice,the weight and index of ovaries of C57BL/6J normal female mice treated with CTX decreased significantly.In addition,after CTX treatment,female mice showed that ovarian tissue atrophy,structural disorder,interstitial necrosis,more atretic follicles and follicular necrosis in cortex,accompanied with fibrosis.And the total number of follicles,primordial,primary,antral follicles and corpus luteum decreased significantly,the number of atretic follicles increased significantly.Among them,the proportion of primordial follicle,antral follicle and corpus luteum decreased significantly,the proportion of atretic follicle increased significantly,the level of FSH increased significantly,the level of AMH and E2 decreased significantly,indicating that CTX induced significant premature ovarian failure.Compared with CTX group,the ovarian tissue of Ang(-/-)+CTX group was further atrophied,the structure was more disordered,the atresia follicle and follicular necrosis were aggravated,the degree of fibrosis was aggravated,the total number of follicles and the number of growth follicles at all levels were further reduced,the number of atresia follicles was significantly increased,but the number of antral follicles and the number of corpus luteum were not significantly affected,the proportion of primary follicles and primary follicles was significantly decreased,The percentage of atretic follicles increased significantly,the level of FSH increased significantly,the level of serum AMH and E2 decreased significantly,and the level of FSH increased significantly,indicating that Ang gene knockout further aggravated the POF induced by CTX.This may be related to Ang gene defects affecting angiogenesis,primordial follicle and all levels of follicular development,promoting follicular atresia,and further affecting hormone secretion. |
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